In eukaryotic cells, DNA synthesis is restricted to a limited interval (S phase) in the cell division cycle. The DNA is functionally divided into many replicating units (replicons) which are duplicated at different but characteristic times within the S phase. Several lines of evidence indicate that protein synthesis is an obligate requirement for continuing DNA synthesis. In this study, we propose to establish a system in tissue culture cells in which we can ask the following question: are specific proteins elaborated at precise times before and during the S period which interact with specific nucleotide sequences in the eukaryotic genome in order to promote replication of these sequences at precise times? We require: 1) a cell line which has at least two marker nucleotide sequences - one which replicates early and the other late in the S period; each marker should be readily identifiable cytologically, and each should be separable from the rest of the genome by physical methods; 2) a method of synchrony in which all cells in the population enter and transverse S with a high degree of synchrony; with such a synchronized population, DNA can be labelled at precise times during S, and proteins can be prepared from the synchronized population at precise times; and 3) an in vitro system in which semi-conservative DNA replication occurs under optimum conditions and which will initiate new chains in vitro upon the addition of appropriate macromolecules. We further propose to examine by polyacrylamide gel electrophoresis the spectrum of proteins elaborated uniquely during the S period which behave in a manner expected of regulatory proteins. Proteins of interest can then be isolated and added to the in vitro system to assess their ability to promote initiation of defined loci.